The Nobel Prize in Physiology or Medicine 1934
George H. Whipple, George R. Minot, William P. Murphy

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Nobel Lecture

Nobel Lecture, December 12, 1934

The Development of Liver Therapy in Pernicious Anemia

The idea that something in food might be of
advantage to patients with pernicious anemia was in my mind in
1912, when I was a house officer at the Massachusetts General
Hospital, as is noted in certain case records there. Ever since
my student days, when I had the opportunity, in my father's wards
at the Massachusetts General Hospital, to distinguish from
pernicious anemia two cases of chronic hereditary hemolytic
jaundice, I have taken a deep interest in this disease. I watched
many patients pass through relapses and remissions, and observed
that, despite treatment with arsenic, blood transfusions,
splenectomy, and other procedures, all eventually died. Prolonged
observation permitted me to become acquainted with the multiple
variations and many aspects of the disease, and to realize that
from a few cases it was difficult to determine the effect of
therapeutic procedures.

The study of the patients' diets was begun
in 1915 in an attempt to determine if some sort 'of dietary
deficiency could be found. The similarity of certain symptoms and
signs of pernicious anemia to those in pellagra, sprue, and
beriberi was appreciated, as was the fact that certain sorts of
anemia were occasionally associated with a faulty diet. Elders,
among others, suggested in 1922 that such a state of affairs
existed in pernicious anemia. Furthermore, the almost constant
occurrence of achlorhydria in pernicious anemia, which appears
usually long before the anemia and remains in spite of liver
therapy, led me to wonder if this disorder of the digestive
system had something to do with the condition which might be in
the nature of a dietary deficiency disease. Indeed, Fenwick,
about 1880, suggested the possible primary role of the stomach,
but it remained for Castle, in 1928, to demonstrate the part this
organ plays in the causation of the disease.

The possibility of an excess of fats in the
diet leading to excessive blood destruction was considered, but
therapy with low-fat diets was futile. Later the effects of
high-caloric: feeding with an excess of protein, derived
especially from meat, were studied at about the same time that
other physicians, such as Barker, reported some benefit from this
treatment. The results were not impressive 'but were perhaps
suggestive.

Although Pepper in 1875 and Cohnheim in
1876 recognized that the bone marrow was abnormal, there was a
prevailing opinion in the early part of this century that
abnormal blood destruction played an important or primary role in
the production of the disease. Nevertheless it was believed by
many physicians, as I was taught, that the production of blood by
the bone marrow was also deeply implicated. About 1919 the late
Dr. James Homer Wright taught me to appreciate the character of
the abnormality of the bone marrow in pernicious anemia, which
led me to believe firmly that something was needed to make the
primitive red cells that crowd the bone marrow in relapse grow to
normal cells; and that it was of no particular value to aim
treatment towards stopping what has been called excessive blood
destruction in this disease. In 1922 Whipple suggested that in
pernicious anemia there might be a scarcity of material from
which the stroma of the red blood cells was formed, or that there
existed a disease of the stroma-forming cells of the bone marrow.
This concept fitted with the idea that there was a deficiency of
something in the body and that dysfunction of pigments metabolism
was resultant, or of secondary importance.

'For centuries the concept that food bore a
relationship to anemia had been vaguely expressed in the
literature. It had been shown that liver and kidneys, rich in
complete proteins, promoted the growth of animals, and that
substances in liver could enhance cell division. It was likewise
recognized that liver-feeding could benefit patients with sprue
(Manson, 1883) and pellagra. These were among the reasons that
led to the choice of liver as a substance likely to enhance blood
formation. Of invaluable importance was Whipple's fundamental and
classical work on hemoglobin regeneration by means of liver and
other foods in anemia due to blood loss in dogs. He has now
placed upon a secure quantitative basis the influence of food
upon anemia.

A few patients were fed relatively small
amounts of liver during 1924 and early 1925. Although these first
patients did better than expected, the results permitted no more
than speculations. Then Dr. Murphy joined in the work and we
pursued the study of these and subsequent cases. Liver had been
fed by Gibson and Howard and other individuals to pernicious
anemia patients but without persistence or definite results. It
seemed to us that to accomplish our object a large weighed amount
of liver should be fed daily with regularity. Likewise to
determine the effect it was considered essential that data should
be obtained in a large number of cases to be appropriately
compared with controls. By May, 1926, we had fed liver
intensively and daily to 45 patients. In many of these patients
symptomatic improvement was obvious within about a week. Soon
they craved food, and color appeared in their faces. Tongue and
digestive symptoms rapidly lessened. Within about 60 days the red
blood cells counts had risen on the average from low levels to
approximately normal. Dr. Murphy will describe to you in more
detail the improvement that takes place. I wish especially to
call to your attention the fact that an objective measure of the
effects upon blood production was the chief basis of our
conclusions that by feeding liver, significant improvement had
been obtained. I refer especially to counts of new adult and
young red blood cells (reticulocytes) appearing, as Peabody's
studies demonstrated later, as a result of the maturation of the
immature cells crowding the bone marrow.

The next step naturally was to attempt to
determine the nature of the constituent in liver responsible for
the effects, and to learn if an extract for therapeutic use could
be obtained. Dr. Edwin J. Cohn, of the Department of Physical
Chemistry in the Laboratories of Physiology of the Harvard
Medical School, soon made a potent extract suitable for oral use.
We tested on patients the preparations he prepared in an attempt
to isolate the active principle. Although unsuccessful in this
objective, as time passed we demonstrated (1929) that the potent
material could be given intravenously, and produced maximal
effects in very small quantities (0.15 g). These small
experimental preparations were not practical for regular use, and
it remained for Gänsslen in Germany to produce the first
practical extract for parenteral therapy. Since then numerous
individuals have prepared and studied such preparations. Extracts
for parenteral use can be easily made by dissolving in water a
powdered extract (Fraction G of Cohn) commonly used in America.
The exact nature of the potent substance remains unknown, but
especially from the studies of Cohn and West it appears to be a
relatively small nitrogenous compound.

Extract given parenterally is at least 50
and perhaps 100 times as potent as extract given by mouth and it
assures the individual of receiving into his body proper the
material he lacks. It thus permits one to give large amounts
easily: e.g., a few cubic centimeters of fluid a week, instead of
about 1,500 to 3,000 grams of liver. The physician must know what
a given amount of a given preparation may be expected to
accomplish under usual circumstances. He must recognize that
extraction causes loss of potency and that the potency of a good
preparation is usually no more than 70 per cent of that of raw
liver. Because one preparation is three times as concentrated as
another, it by no means follows that the amount of potent
material contained is three times as great. It may actually be
less potent. Active material is not confined to liver. It has
been found to occur in stomach tissue by Sturgis and Isaacs, and
is found in kidney, brain, placenta, and probably other organs.
Castle has shown that a reaction between an unidentified
substance in normal gastric juice and material in certain foods
rich in the vitamin B complex yields potent material. By
utilization of the principle described by Castle, Reimann has
shown that liver may have its potency increased by incubation
with gastric juice. This has resulted in the practical use of
combinations of gastric and liver tissue.

Since the presentation of our original
work, the regular beneficial results of liver therapy have been
confirmed in many parts of the world; for example, in Sweden by
Strandell and in Copenhagen by Meulengracht. There have been
differences of opinion regarding the value of one or another
preparation, but these differences can largely be explained if a
proper comparison of dosage is made. There are cases requiring
much more potent material than others and in some cases there is
difficulty of absorption, making parenteral therapy essential. As
in other deficiency disorders there are factors, such as
infections, arteriosclerosis and serious damage to vital organs,
which inhibit the action of "liver extract" and when they are
present it is often necessary to give unusually large amounts of
the active principle. The results of treatment will be
essentially the same if, irrespective of its source for the given
case, enough potent material enters the body throughout life.
Indeed, pernicious anemia, like other deficient states, should be
treated on a quantitative basis. Failure of liver therapy in a
case diagnosed pernicious anemia implies inadequate treatment, an
incorrect diagnosis, or the existence of complication
sufficiently serious in itself to be disastrous for the
patient.

The treatment should not consist in
supplying enough material to remove only one symptom, such as
anemia, but enough both to supply indefinitely all demands of the
body for the substance and to fill it with an adequate reserve
supply. The disease affects the gastro-intestinal and neural
systems as well as the hemopoietic tissue. Probably more material
is required to improve or inhibit the progress or development of
neural lesions than to permit blood element to be maintained.
With proper dosage, symptoms due to neural lesions usually
lessen, sometimes strikingly, as Dr. Murphy and I originally
noted. Unfavourable reports concerning the effects on the neural
system can be attributed to failure to realize that there is no
standard dose of "liver extract", and that there is great
variation in the potency of different preparations; also that
sepsis and other complications can inhibit the action of liver on
the nervous tissue, as it does on blood formation. For proof of
the effect of liver on neural lesions one must not so much study
the degree of improvement, as seek for evidence of the arrest of
the process. In my clinic this has been done especially by Dr.
Strauss. There have been observed critically over from two to
three years about 100 cases, including 26 cases with advanced
combined system-disease, treated parenterally with liver extract.
In no instance did a single objective neurologic sign become more
marked nor did an abnormal sign develop. Abnormal signs sometimes
decreased in intensity or became normal. There was thus objective
arrest of the neural lesions in every case and subjective
improvement in all. Indeed, certain patients who were originally
unable to walk became sufficiently improved to return to their
occupations. Failure to arrest the progress of neural lesions,
like failure to restore the blood to normal and keep it there,
usually means that more liver is required. Sometimes such severe
infection or the like is present, that arrest cannot be achieved.
One should not interpret parenteral therapy as acting in any
fundamentally different manner from oral therapy. Certain cases
treated for over eight years orally have had no progress of their
neural lesions. Many cases can be adequately treated orally, but
parenteral therapy is simple and permits suitable amounts of
active principle easily and assuredly to enter the patient's
body.

Liver therapy has been shown to be of value
in other anemias than the idiopathic pernicious anemia originally
described by Addison in 1849. There are other substances in liver
and in certain liver extracts potent for pernicious anemia
besides the principle active in pernicious anemia. Dr. Whipple
has demonstrated a factor in liver aiding blood regeneration in
"secondary a anemia. This factor can affect blood formation in
certain clinical cases but as yet has received little critical
study in man. Liver extract potent for pernicious anemia is of
value in such other macrocytic anemias as arise in sprue
(Bloomfield and Wycoff, Ashford and others), pregnancy (Wills,
Strauss, and Castle), coeliac disease (Vaughan et al.) and
the like. Partly because we recognized in pernicious anemia that,
in addition to improvement of the blood, the alimentary tract and
nervous systems were benefited, a still wider application has
resulted. The possibility of controlling the gastro-intestinal
symptoms in sprue by the use of parenteral therapy with liver
extract has been demonstrated by Castle and Rhoads. For this
purpose it is of great value. Likewise in pellagra the
gastro-intestinal symptoms are responsive. The improvement of the
skin lesions in this disease, which also occurs, suggests the
possibilities of a broader application to other conditions. It
was suggested, in 1913, by Vogel and McCurdy, that determinations
of the numbers of the young red blood cells (reticulocytes) might
measure within a brief time the effect of therapy. Although I had
studied these cells from my student days in many patients, it was
not until Dr. Murphy's and my observations were well under way,
that the significance of the reticulocyte reaction as an index of
the effect of potent material was fully appreciated. It then
became an important aid in the evaluation of therapy and in the
subsequent development of effective liver extracts. It was soon
recognized that, following the administration of effective doses
of liver, the reticulocytes in pernicious anemia are increased
for a few days, during which time their numbers follow a
distinctive course. This reticulocyte reaction is orderly and
simulates the curve for growth and death of organisms and their
cells. Critical daily observation of these reactions and their
proper interpretation thus gives useful information concerning
both the state of the patient and the potency of the material
administered.

A reaction that does not conform in
character to that due to liver and in proper time relation to the
administration of the substance under test, should always be
looked upon with suspicion. The reticulocyte reaction induced by
liver indicates a bone marrow reaction to a physiological need
and not to a stimulant: a normal reaction to the existing anemia.
A reticulocyte response does not necessarily mean that specific
material which the body lacks has been supplied, for there are
other substances and conditions that cause reticulocytosis in
pernicious anemia, which do not regularly promote normal blood
formation. For example, responses may be induced by potassium
arsenite (Fowler's solution), but they usually differ very much
from the physiological responses to liver. Non-specific
responses, however, may closely resemble those due to supplying
the deficient substance. One must be particularly critical of the
nature of responses caused by substances given parenterally,
because non-specific responses of various sorts can arise more
readily from parenteral injection than from feeding.

It is important that the potency of
products used in life-saving procedures be assured. The
reticulocyte reaction is serviceable in yielding information
regarding the strength of liver extracts and potent substitutes
employed clinically. The factors which physiologically influence
the reticulocyte reaction, also influence the rate of total red
cell regeneration; but, as noted in 1927, the amount of material
necessary to induce maximal reticulocyte rises is often less than
the amount necessary for the maximal rate of increase of red
cells. In testing products one must aim to give an amount
expected to yield less than a maximal reticulocyte response,
since otherwise considerable losses in potency may occur and
remain undetected. The percentage of reticulocytes at the peak of
their rise for a given red cell level has been used in
comparative data. It is, of course, difficult to determine
whether one agent is more effective than another, unless tests
have been made in the same way. Very different types of curves
for reticulocytes will be obtained from daily administration and
from one relatively large parenteral injection, so that
reticulocyte peaks cannot be compared directly. In either
instance, however, the total number of young red cells poured
forth during the reaction will be similar.

Because of the considerable variation in
the reactivity of cases, more information can undoubtedly be
obtained by comparative tests of known and unknown material in
the same patient. This can be accomplished by observing the
reticulocyte responses in successive ten-day periods of uniform
daily administration of each substance. The dosage of the
substance of known potency given first must be such that a
submaximal reticulocyte response will result. If so much material
has been given that a maximal reticulocyte response has occurred,
an increase of potent material in the second period cannot induce
a second reticulocyte rise. In a properly conducted test the
occurrence of any orderly reticulocyte response to a second
substance means that it is of greater potency than the first
material given.

There is need for an animal or laboratory
procedure to test the potency of products and to aid in
determining the nature of the substance or substances effective
in pernicious anemia. Numerous studies of this sort have been
made; for example, by Vaughan on pigeons. Jacobson's recent
report concerning the use of reticulocyte reactions in guinea
pigs is suggestive of fruitful results.

In the early days, as has been noted, it
was thought that pernicious anemia might be a dietary deficiency
disease. The demonstration of the effectiveness of liver therapy
indicated that this was very probably the case and thus provided
the proper orientation towards a study of its cause. Castle in my
clinic, from work entirely his own, has shown that it is a
deficiency disease of a special sort, one which may be spoken of
as a conditioned dietary deficiency disease. He and his
associates have demonstrated that because of a specific defect of
the gastric secretion the patient with pernicious anemia is
unable to carry out an essential reaction with certain
constituents of food. In the normal individual this reaction is
necessary for the production of the supply of "liver extract" and
thus for the prevention of the disease. The material is absorbed
and stored in the liver and certain other tissues, so that when
such animal tissues are fed to human beings, they receive the
material needed in pernicious anemia without the necessity of the
special reaction within their stomachs. In pernicious anemia it
has been shown (Ivy and others) that "liver extract" is absent in
the patient's liver. The factor in the normal gastric secretion
responsible for the reaction has not been identified with any of
its recognized constituents. Meulengracht has shown from what
portion of the stomach the gastric factor is formed in pigs.
Strauss and Castle have demonstrated that the food factor is
associated with a number of natural sources of vitamin B,
although, from the work of Wills, Lassen and others, it is
probable that it is not a portion of the vitamin B complex.

As a logical consequence of this work
Castle and his associates have postulated the significance of
defects of the dietary factor, the gastric factor, and of
difficulty in the absorption or utilization of substances
promoting blood formation in the production of other types of
macrocytic anemia responding to liver extract. Thus, in certain
instances of the tropical macrocytic anemia of sprue (Castle and
Rhoads) and in the anemia of pregnancy studied by Wills in India,
a dietary defect seems of primary importance. The macro-cytic
anemia resulting from total ablation of the stomach or its
destruction by' cancer, like Addisonian pernicious anemia, is
especially associated with loss of the gastric factor. In the
macrocytic anemia of certain cases of late sprue, of coehac
disease and of rare instances of intestinal stenosis or multiple
anastomoses, difficulty in absorption plays a major role. In the
anemia of fish-tapeworm infestation these factors may also be
involved. Theoretically there could occur a disorder of the
internal metabolism of "liver extract", and an occasional case of
macrocytic anemia might be explained on such an assumption. Today
we realize also that the participation of these factors may be
variable and temporary, as occurs in the pernicious anemia of
pregnancy of the temperate zone (Strauss and Castle). During
pregnancy the gastric factor may be absent, only to reappear
after delivery. Macrocytic anemia in animals has been produced by
Wills, and by Rhoads and Miller as a result of dietary defects,
and Bence has shown that the liver of gastrectomized pigs becomes
deficient in "liver extract".

In man, however, dietary deficiency is
seldom confined strictly to one factor, nor are the results of
disturbances of gastric secretion, of defects of intestinal
absorption or of utilization necessarily concerned in only one
type of metabolic process. Clearly such disturbances are involved
in the production of iron deficiency resulting in certain types
of hypochromic anemia. We have noted that combined deficiency of
iron and "liver extract" is not rare in the same individual.
Other double and even multiple deficiencies occur. In one rare
and striking case I have seen the tongue and skin lesions of
pellagra subside upon the oral administration of yeast, the edema
from protein deficiency vanish when beefsteak was fed and finally
the macrocytic anemia disappear rapidly when liver extract was
given orally.

Castle and his associates have shown that the gastric factor may
return toward normal in pernicious anemia after treatment with
liver extract. It is probable that the gastric reaction proceeds
somewhat according to the law of mass action, so that very little
of the intrinsic factor of the stomach might produce with a large
amount of the extrinsic factor of the food, and vice versa,
material for absorption sufficient to meet to a significant
degree the demands of the body. Such a state of affairs could
explain the responses in pernicious anemia obtained by feeding
large amounts of autolyzed yeast-extract, as shown, for example,
by Ungley. Here it is probable that traces of the intrinsic
factor were present; and Isaacs, Goldhamer, and Sturgis have
shown that traces, rather than complete absence, of this factor
are apt to occur in Addisonian pernicious anemia. In this
disease, however, it is almost always the gastric factor that is
grossly deficient rather than the dietary factor; but both
substances may be involved. We have seen patients who formerly
had satisfactory diets, develop pernicious anemia lasting many
months or a few years after partaking of distinctly undesirable
diets. In such instances the poor diet probably precipitated the
onset of the disease. It is thus not difficult, in the light of
modern knowledge, to understand the probable causation of the
"spontaneous" remissions and relapses in pernicious anemia.
Probably in the natural course of the disease the gastric factor
slowly declines but fluctuates in quantity from time to time. The
extrinsic factor will also vary, and certain foods and rest may
stimulate an increase of the intrinsic factor. Infection and
severe gastritis could be responsible for temporary decrease of
the gastric factor. The occasional patient who, after a relapse,
remains without liver therapy in a state of complete remission
for years, probably represents an instance of a temporary marked
decline in the gastric factor, unless grossly deficient diet is
shown to have existed.

Patients undergoing liver therapy for
pernicious anemia sometimes, of their own accord, omit treatment
and may remain in apparent good health for many months, but many
more of such individuals soon find they are sick again. The
former perhaps have accumulated a considerable reserve supply of
"liver extract" in their bodies and have probably been enabled by
treatment to manufacture some potent material from an ordinary
diet. Sooner or later a very large number of these patients will
relapse, if proper treatment is not resumed. The grave error in
treatment is to prescribe too little liver extract or potent
substitute. When there is doubt, more rather than less should be
given. It is essential that the individual receive into his body
indefinitely and with regularity enough potent material for his
given case. The physician, however, must do more for his patient
than prescribe a proper amount of liver, stomach, or the like; he
should attend to all aspects of the case and not neglect
attention to the individual's manifold problems of thought and
action.

I have attempted to outline Dr. Murphy's
and my contribution to the work for which the Caroline Institute
has honored us. I have pointed out to you, also, some of the
studies that have been made as the result of demonstrating with
Dr. Murphy that liver feeding is dramatically effective for
pernicious anemia patients. It seems to me that one may expect in
the future more information to be obtained which, directly or
indirectly, will follow as the result of these observations.
Thus, upon the foundations laid by previous investigators, do
medical art and science build a structure which will in its turn
be the foundation of future knowledge.